Heparin affinity tag and applications thereof

ABSTRACT

In one aspect, affinity tags for recombinant protein purification are described herein which, in some embodiments, can mitigate or overcome disadvantages of prior affinity tag systems. In some embodiments, for example, affinity tags described herein permit efficient elution of desired recombinant proteins with simplified solution systems, such as alkali metal salt solutions. An affinity tag described herein comprises an amino acid sequence including a repeating amino acid unit of BXXXBXX, wherein B is an amino acid selected from the group consisting of histidine, lysine and arginine and X is an amino acid selected from the group consisting of amino acids other than histidine, lysine and arginine.

FIELD

The present invention relates to affinity tags for protein purificationand, in particular, to affinity tags binding heparin.

BACKGROUND

Overexpression and purification of recombinant proteins are ofsignificant interest to the pharmaceutical and biochemical industries.Recombinant proteins, for example, are used in a variety of commerciallyimportant applications, including therapeutics, bioinsecticides,diagnostic kits and many others. Advances in recombinantdeoxyribonucleic acid (DNA) technology and protein expression systemshave rendered practical the production of proteins in significantquantities employing a variety of hosts. However, rapid and efficientpurification of recombinant proteins remains a major challenge.Downstream purification of recombinant proteins can account for about80% of total production cost. Therefore, cost-effective purificationmethods are of critical importance to pharmaceutical and biotechnologycompanies.

In this context, affinity chromatography is the method of choice forprotein purification. This method involves addition of a selectiveaffinity tag sequence to the target protein gene to generate the fusiongene. The fusion protein, produced by overexpression of the fusion genein heterologous hosts, is purified by exploiting the highly specificbinding characteristics of the affinity tag. The affinity tag issubsequently removed from the target recombinant protein. Severalaffinity tags such as polyhistidine, glutathione S-transferase, maltosebinding protein, chitin, thioredoxin, small ubiquitin modifier protein(SUMO), N-utilization substance A (Nus A) and others have been popularlyemployed for recombinant protein purification. Nevertheless, commonproblems afflict these affinity tags, including decreased columncapacity due to large molecular size of the affinity tags, high cost ofelution solutes, tendency of the affinity tag(s) to be expressed asinsoluble inclusion bodies, problems in recovery of cleaved affinitytags and difficulties in accurately maintaining the pH of solution usedfor protein elution.

SUMMARY

In one aspect, affinity tags for recombinant protein purification aredescribed herein which, in some embodiments, can mitigate or overcomedisadvantages of prior affinity tag systems. In some embodiments, forexample, affinity tags described herein permit efficient elution ofdesired recombinant proteins with simplified solution systems, such asalkali metal salt solutions. An affinity tag described herein comprisesan amino acid sequence including a repeating amino acid unit of BXXXBXX,wherein B is an amino acid selected from the group consisting ofhistidine, lysine and arginine and X is an amino acid selected from thegroup consisting of amino acids other than histidine, lysine andarginine. In some embodiments, for example, the amino acid sequence ofthe affinity tag includes the sequence of SEQ ID NO:1 or SEQ ID NO:2.Alternatively, an affinity tag described herein comprises an amino acidsequence selected from Table I, wherein B is an amino acid selected fromthe group consisting of histidine, lysine and arginine and X is an aminoacid selected from the group consisting of amino acids other thanhistidine, lysine and arginine. Further, an affinity tag describedherein, in some embodiments, selectively binds heparin.

Table I  Amino acid sequences of affinity tags XBXXBXXBXXBX XBXXBXXBXXBXBXBXXBXBXBXB BBBBXXBBB XBBBXXBBBX XBBBXXXXBBBXXXXBBBX XBXBXXBXBXXBXBXXBXXBXBXXBXBXXBX XBBBXXBBBX XBXBXXXXXBXBX XBXXBXXBX XBBXBXXXBXXXBXBXBXBXXXXBXXXBX XBXXXBXBBBXBXXBXXXBX XBXXXBXXBXXBX XBBXXBXBBBXXBBXBXBXXXBX XBBXBXBBX XBXXBXBBXXBX XBXBXXXXBXXXBX XBBXXBXBBBX

In another aspect, recombinant protein compositions are described hereinemploying affinity tags for purification processes. A recombinantprotein composition described herein comprises a first amino acidsequence of a target protein and an affinity tag coupled to the firstamino acid sequence, the affinity tag comprising an amino acid sequencefor binding heparin. The amino acid sequence of the affinity tag cancomprise a repeating amino acid unit of BXXXBXX, wherein B is an aminoacid selected from the group consisting of histidine, lysine andarginine and X is an amino acid selected from the group consisting ofamino acids other than histidine, lysine and arginine. For example, theamino acid sequence of the affinity tag can include the sequence of SEQID NO:1 or SEQ ID NO:2. Moreover, in other embodiments, the amino acidsequence of the affinity tag can comprise an amino acid sequenceselected from Table I. Further, a protease cleavage site can bepositioned between the first amino acid sequence of the target proteinand the affinity tag amino acid sequence. In some embodiments, asuitable protease cleavage site is a serine protease cleavage site orcysteine protease cleavage site.

Recombinant DNA encoding protein compositions described herein are alsoprovided. A recombinant DNA molecule, for example, encodes an amino acidsequence of a target protein and associated affinity tag amino acidsequence for binding heparin. The encoded amino acid sequence forbinding heparin comprises a repeating amino acid unit of BXXXBXX,wherein B is an amino acid selected from the group consisting ofhistidine, lysine and arginine and X is an amino acid selected from thegroup consisting of amino acids other than histidine, lysine andarginine. In some embodiments, the recombinant DNA molecule comprisesthe nucleotide sequence of SEQ ID NO:3 or SEQ ID NO:4 encoding the aminoacid sequence of the affinity tag. Additionally, in some embodiments,the encoded amino acid sequence for binding heparin comprises an aminoacid sequence selected from Table I.

In another aspect, methods of purifying recombinant protein compositionsare described herein. A method of purifying a recombinant proteincomposition comprises providing a mixture including the recombinantprotein composition, the recombinant protein composition comprising afirst amino acid sequence of a target protein and an affinity tagcoupled to the first amino acid sequence, the affinity tag comprising anamino acid sequence for binding heparin. The mixture is loaded onto aseparation column having a stationary phase comprising heparin, and therecombinant protein is bound to the heparin stationary phase via theaffinity tag. The bound recombinant protein is subsequently eluted fromthe column. In some embodiments, the mixture comprising the recombinantprotein composition is a lysate, such as that from the expressing host.

As described herein, the affinity tag amino acid sequence for bindingheparin can comprise a repeating amino acid unit of BXXXBXX, wherein Bis an amino acid selected from the group consisting of histidine, lysineand arginine and X is an amino acid selected from the group consistingof amino acids other than histidine, lysine and arginine. Additionally,in some embodiments, the amino acid sequence for binding heparincomprises an amino acid sequence selected from Table I. Further, aprotease cleavage site can be positioned between the first amino acidsequence of the target protein and the affinity tag amino acid sequence.Therefore, the affinity tag can be cleaved from the recombinant proteinat the cleavage site with suitable protease.

In a further aspect, separation media are described herein which can beused in the purification of protein compositions. A separation mediumdescribed herein comprises a support phase and a stationary phaseattached to the support phase, the stationary phase including an aminoacid sequence comprising a repeating amino acid unit of BXXXBXX, whereinB is an amino acid selected from the group consisting of histidine,lysine and arginine and X is an amino acid selected from the groupconsisting of amino acids other than histidine, lysine and arginine. Insome embodiments, for example, the amino acid sequence comprises thesequence of SEQ ID NO:1 or SEQ ID NO:2. In other embodiments, thestationary phase can include an amino acid sequence selected from TableI. The amino acid sequence of the stationary phase can be operable toselectively bind heparin. Additionally, the amino acid sequence can beattached to the stationary phase through a spacer.

Methods of purifying heparin are also described herein. A method ofpurifying heparin comprises providing a mixture including heparin andloading the mixture onto a separation column comprising a support phaseand a stationary phase attached to the support phase, the stationaryphase including an amino acid sequence comprising a repeating amino acidunit of BXXXBXX, wherein B is an amino acid selected from the groupconsisting of histidine, lysine and arginine and X is an amino acidselected from the group consisting of amino acids other than histidine,lysine and arginine. In some embodiments, the stationary phase aminoacid sequence comprises the sequence of SEQ ID NO:1 or SEQ ID NO:2.Alternatively, the stationary phase can include an amino acid sequenceselected from Table I. Heparin from the mixture is bound to thestationary phase and subsequently eluted from the column.

In a further aspect, polyclonal antibodies are provided for recognizingaffinity tags described herein. In some embodiments, a compositioncomprises polyclonal antibodies recognizing the amino acid sequence ofSEQ ID NO:10. For example, the polyclonal antibodies can be bound to anantigen comprising an amino acid sequence incorporating SEQ ID NO:10.Moreover, the antigen can be an affinity tag further comprising arepeating amino acid unit of BXXXBXX, wherein B is an amino acidselected from the group consisting of histidine, lysine and arginine andX is an amino acid selected from the group consisting of amino acidsother than histidine, lysine and arginine. Additionally, the affinitytag can be coupled to a target protein as described herein.

These and other embodiments are described further in the detaileddescription which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a pep-wheel diagram of an affinity tag according to oneembodiment described herein.

FIG. 2 illustrates a synthetic pathway for a plasmid encoding a targetprotein and associated heparin binding (HB) affinity tag according toone embodiment described herein.

FIG. 3 is a flow chart illustrating a synthetic pathway for a plasmidencoding a target protein and associated heparin binding (HB) affinitytag according to one embodiment described herein.

FIG. 4 illustrates an elution profile of bound and unbound fractions inresponse to a NaCl gradient according to one embodiment describedherein.

FIG. 5 is a Western blot of protein fractions eluted from a heparinsepharose column using a stepwise salt gradient according to oneembodiment described herein.

FIG. 6 is a dot blot demonstrating limits of detection of a targetprotein and associated heparin binding affinity tag according to anembodiment described herein.

FIG. 7 provides mass spectrometry results identifying (A) the targetprotein and (B) heparin binding affinity tag according to one embodimentdescribed herein.

FIG. 8 is an isothermogram depicting titration of the HB affinity tagwith heparin.

FIG. 9 is a far UV circular dichroism (CD) spectrum of a purified targetprotein according to one embodiment described herein.

FIG. 10 are far UV CD spectra of a heparin binding affinity tag in anunbound state and a heparin bound state according to one embodimentdescribed herein.

DETAILED DESCRIPTION

Embodiments described herein can be understood more readily by referenceto the following detailed description and examples and their previousand following descriptions. Elements, apparatus and methods describedherein, however, are not limited to the specific embodiments presentedin the detailed description and examples. It should be recognized thatthese embodiments are merely illustrative of the principles of thepresent invention. Numerous modifications and adaptations will bereadily apparent to those of skill in the art without departing from thespirit and scope of the invention.

I. Affinity Tags

In one aspect, affinity tags for recombinant protein purification aredescribed herein. An affinity tag described herein comprises an aminoacid sequence including a repeating amino acid unit of BXXXBXX, whereinB is an amino acid selected from the group consisting of histidine,lysine and arginine and X is an amino acid selected from the groupconsisting of amino acids other than histidine, lysine and arginine Insome embodiments, for example, X is an amino acid selected from TableII.

TABLE II Amino Acids of X Alanine Asparagine Aspartic Acid CysteineGlutamic Acid Glutamine Glycine Isoleucine Leucine MethioninePhenylalanine Proline Serine Threonine Tryptophan Tyrosine ValineThe amino acid unit of BXXXBXX, in some embodiments, repeats at leastthree times in the affinity tag amino acid sequence. The amino acid unitcan repeat sequentially or amino acid(s) can be positioned between theindividual repeating amino acid units. For example, an affinity tag caninclude the generic sequence of -XXBXXBXXXBXXBXXXBXXBXXXBXXBXXBXX-,wherein X and B are defined above. Alternatively, an affinity tagdescribed herein comprises an amino acid sequence selected from Table I.Selection of specific amino acids for X and B can be governed by theintended functionality of the amino acid sequence in the affinity tag.For example, the amino acid sequence can be employed to bind heparin. Insuch embodiments, X and B can be selected to produce a peptide structurehaving high propensity to form an amphipathic helix, wherein side chainsof the polar and non-polar amino acids are oriented on opposite sides ofthe helix. Moreover, polar amino acids of the sequence should havesuitable locations for forming electrostatic interactions with heparin.Further, more than about 60% of the amino acids chosen for the genericsequence should be polar to facilitate overexpression ofrecombinant-affinity tag proteins in the soluble form. In view of theseconsiderations, the generic amino acid sequence, in some embodiments,can have the specific sequence of SEQ ID NO:1 or SEQ ID NO:2. FIG. 1 isa pep-wheel diagram of the amino acid sequence of SEQ ID NO:1illustrating the foregoing design principles for heparin binding. Asillustrated in FIG. 1, the resulting peptide displays an asymmetricdistribution of the polar and non-polar amino acids, wherein N and Crepresent the N- and the C-terminal ends of the peptide.

As provided further herein, affinity tags having amino acid sequencesand structure described in this Section I can be expressed fromrecombinant DNA encoding for the sequence. For example, SEQ ID:3 is thenucleotide sequence encoding the amino acid sequence of SEQ ID NO:1.Similarly, SEQ ID:4 is the nucleotide sequence encoding the amino acidsequence of SEQ ID NO:2. Alternatively, affinity tags having amino acidsequences described herein can be synthesized by solid-state methodsknown to those of skill in the art. Solid-state synthesis of theaffinity tags can find applicability in separation media describedbelow.

II. Recombinant Protein Compositions and Recombinant DNA

In another aspect, recombinant protein compositions are described hereinemploying affinity tags for purification processes. A recombinantprotein composition described herein comprises a first amino acidsequence of a target protein and an affinity tag coupled to the firstamino acid sequence, the affinity tag comprising an amino acid sequencefor binding heparin. The affinity tag can employ any amino acid sequencesuitable for binding heparin. In some embodiments, the affinity tagcomprises an amino acid sequence described in Section I above. Forexample, the affinity tag can include an amino acid sequence comprisinga repeating amino acid unit of BXXXBXX, wherein B and X are defined inSection I herein. In particular embodiments, the affinity tag includesthe sequence of SEQ ID NO:1 or SEQ ID NO:2. Moreover, in otherembodiments, the amino acid sequence of the affinity tag can comprise anamino acid sequence selected from Table I.

Further, a protease cleavage site can be positioned between the firstamino acid sequence of the target protein and the affinity tag aminoacid sequence. Any protease cleavage site or restriction site notinconsistent with the objectives of the present invention can be usedfor cleavage of the affinity tag from the recombinant target protein. Insome embodiments, a suitable protease cleavage site is designed forserine protease(s). A serine protease cleavage site, for example, may beconstructed for use with thrombin. SEQ ID NO:5 and SEQ ID NO:6 eachprovide a specific affinity tag amino acid sequence coupled to athrombin cleavage site. Alternatively, a cleavage site can beconstructed for cysteine protease(s). A cysteine protease cleavage site,for example, may be constructed for use with tobacco etch virus (TEV)protease. Other suitable protease cleavage or restriction sites caninclude factor Xa, enterkinase and peptides modified with cyanogenbromide. Identity of cleavage site is not a limiting factor foremployment of affinity tags described herein.

Recombinant DNA encoding the target protein and associated affinity tagis inserted into a host for overexpression. The recombinant DNAmolecule, for example, can encode any affinity tag amino acid sequencedescribed in Section I above. In some embodiments, the recombinant DNAmolecule encodes an affinity tag amino acid sequence comprising arepeating amino acid unit of BXXXBXX, wherein B is an amino acidselected from the group consisting of histidine, lysine and arginine andX is an amino acid selected from Table II herein. The recombinant DNAmolecule can encode for the amino acid unit to be repeated at leastthree times in the affinity tag sequence. In a particular embodiment,the recombinant DNA encodes an affinity tag amino acid sequence of SEQID NO:1. In such an embodiment, the recombinant DNA molecule includesthe nucleotide sequence of SEQ ID NO:3. In another embodiment, therecombinant DNA can encode an affinity tag amino acid sequence of SEQ IDNO:2 and include a nucleotide sequence of SEQ ID NO:4. Additionally, insome embodiments, the recombinant DNA molecule encodes an affinity tagamino acid sequence comprising a sequence selected from Table I.Recombinant DNA encoding affinity tags described herein can beartificially synthesized according to techniques known to one of skillin the art.

In addition to the target protein and the affinity tag, the recombinantDNA molecule may encode for a protease cleavage site between the targetprotein and affinity tag. As described herein, a serine or cysteineprotease cleavage site can be employed for cleavage of the affinity tagsubsequent to purification of the recombinant protein. For example, therecombinant DNA molecule can encode the amino acid sequence of SEQ IDNO:5. In such an embodiment, the recombinant DNA molecule comprises thenucleotide sequence of SEQ ID NO:7. Alternatively, the recombinant DNAmolecule can encode the amino acid sequence of SEQ ID NO:6, therebyincluding the nucleotide sequence of SEQ ID NO:8.

Various vectors encoding the target protein, associated affinity tag andprotease cleavage site can be employed. For example, plasmids or viralvectors can be used. Alternatively, yeast or mammalian vectors can beused. Appropriate amplification techniques and restriction enzymes areused to construct vectors for expression of the target protein andassociated affinity tag by a host. Moreover, the vector can includecontrol sequences such as promoter sequences. Expression controlsequences of the vector can vary depending on whether the vector isdesigned to express a nucleotide sequence in a prokaryotic or eukaryotichost. Expression control sequences may include transcriptionalregulatory elements such as promoters, enhancer elements andtranscriptional termination sequences, and/or translational regulatoryelements, such as translational initiation and termination sites. FIG. 2illustrates construction of a vector encoding a target protein andassociated heparin binding affinity tag according to an embodimentdescribed herein. As illustrated in FIG. 2, plasmid pET28a (or plasmidpET22b) containing the affinity tag for binding heparin is digested withrestriction enzymes Barn HI and XhoI. Genes containing the targetprotein are also digested with restriction enzymes Barn HI and XhoI. Thegene inserts subsequently undergo ligation with the doubly digestedpET28a (or pET22b) vector to complete vector construction. As describedherein, the vector can also include a protease cleavage site between theaffinity tag and target protein.

Once the vector encoding the target protein, affinity tag and proteasecleavage site is prepared, the vector is introduced into an appropriatehost cell by any of a variety of suitable techniques, includingtransformation as known in the art. In some embodiments, for example,the constructed vector is inserted into bacterial host(s) such asEscherichia coli. After transformation, recipient cells are grown in anappropriate medium and overexpression of the target protein isadministered.

III. Recombinant Protein Purification

In another aspect, methods of purifying recombinant protein compositionsare described herein. A method of purifying a recombinant proteincomposition comprises providing a mixture, such as a lysate, includingthe recombinant protein composition, the recombinant protein compositioncomprising a first amino acid sequence of a target protein and anaffinity tag coupled to the first amino acid sequence, the affinity tagincluding an amino acid sequence for binding heparin. In someembodiments, the affinity tag comprises an amino acid sequence describedin Section I above. For example, the affinity tag can include an aminoacid sequence comprising a repeating amino acid unit of BXXXBXX, whereinB and X are defined in Section I herein. In particular embodiments, theaffinity tag includes the sequence of SEQ ID NO:1 or SEQ ID NO:2.Moreover, the affinity tag can include an amino acid sequence selectedfrom Table I.

The mixture is loaded onto a separation column having a stationary phasecomprising heparin and the recombinant protein is bound to the heparinstationary phase via the affinity tag with the unbound fraction passingthrough the column. The bound recombinant protein is subsequently elutedfrom the column. Elution of the recombinant protein, in someembodiments, is achieved with an alkali metal salt gradient. Forexample, a sodium chloride (NaCl) gradient can be used for elution ofthe bound recombinant protein. Other suitable salt eluents notinconsistent with the objectives of the present invention are alsocontemplated. The eluted recombinant protein can demonstrate purity inexcess of 90%. In some embodiments, the eluted recombinant protein is atleast 95% pure.

A cleavage site is positioned between the heparin binding affinity tagand target protein. A serine or cysteine protease cleavage site, forexample, can be employed for cleavage of the affinity tag fromrecombinant protein subsequent to protein purification. For example, athrombin cleavage site as set forth in sections of amino acid sequencesSEQ ID NO:5 and SEQ ID NO:6 can be used. The heparin binding affinitytag is cleaved from the recombinant protein, and the resulting mixtureis loaded to a separation column comprising heparin stationary phase.The affinity tag is bound to the heparin stationary phase and therecombinant protein passes through the column as a purified unboundfraction.

EXAMPLE

Bacterial expression vector pET28a was modified as follows to includethe nucleotide sequence of SEQ ID NO:7 encoding a heparin binding (HB)affinity tag and thrombin cleavage site and the nucleotide sequence ofSEQ ID NO:9 encoding the C2A domain of rat synaptotagmin. SEQ ID NO:7was cloned into pET28a, and the resulting plasmid was isolated using theQiagen Mininprep Plasmid Purification kit. Approximately 5 μg of plasmidwas obtained from 3 mL of bacterial culture that was incubated overnightat 37° C. under constant stirring conditions (250 rpm) with suitableantibiotic. Using gene specific primers with the desired restrictionsites (BamHI and Xhol), genes corresponding to C2A (SEQ ID NO:9),S100A13 and C-termAlb3 were amplified using the Phusion PCR Mastermixwith an appropriate DNA template. Polymerase chain reaction (PCR)conditions employed were consistent with the protocol provided by thevendor. Annealing temperature for optimal primer binding wasappropriately set for each clone (62.5° C. for C2A, 64° C. for S100A13and 62.4° C. for C-termAlb3). After successful amplification of the geneinserts, PCR products were subjected to cleanup using the Qiagen PCRcleanup kit. Further, double digestion of the inserts and HB modifiedpET28a plasmid was administered with BamHI and Xhol. The double-digestedpET28a plasmid was subjected to dephosphorylation reaction usingAntarctic phosphatase enzyme. The dephosphorylated pET28a plasmid anddouble digested inserts, including C2A, were resolved on 1% agarose gelfollowed by gel extraction and elution. Ligation of the C2A insert andHB modified pET28a plasmid was subsequently performed using T4 ligase at22° C. for 20 minutes. The ligation product was used to carry-outtransformation of chemically competent DH5α cells, which were plated onagar plates with suitable antibiotic. After an incubation period of14-16 hours, transformations observed on the plates were subjected toscreening by colony PCR method using Taq polymerase. Plasmids frompositive colonies were then isolated and confirmed both by doubledigestion and DNA sequencing.

FIG. 3 is a flow chart illustrating the foregoing synthesis. Further, asillustrated in FIG. 2, bacterial vector pET22b can also be modified toinclude the nucleotide sequence of SEQ ID NO:7 encoding a HB affinitytag and thrombin cleavage site and the nucleotide sequence of SEQ IDNO:9 encoding the C2A domain of rat synaptotagmin. Use of pET28a andpET22b permits selection of the antibiotic resistance[ampicillin(pET22b) or kanamycin(pET28a)] and also the frame for cloninggenes in different multiple cloning sites (MCS).

Overexpression

The pET28a plasmid encoding the heparin binding affinity tag (HB),thrombin cleavage site and C2A domain of rat synaptotagmin wastransformed into BL21(DE3) cells. Overexpression of the HB-fused C2Atarget protein was administered in LB medium. Bacterial cells wereallowed to grow in a suitable antibiotic medium until the OD reached0.4-0.5. Upon reaching the desired OD, cells were induced with 1 mM IPTGand were allowed to grow for an additional time period of 4 hours. Cellswere later harvested by centrifugation under refrigerated conditions (at4° C.) at 6000 rpm.

Purification

Overexpressed bacterial cells were lysed by subjecting the cells tothree passes through the French Cell Press followed by 10 cycles ofsonication (with 10 seconds each in the on-phase and off-phase in icewith an amplitude of 3 and 11 watts output). Overexpression of theHB-fused C2A (HB-C2A) revealed that the protein was predominantlyexpressed in the soluble form in the supernatant of the E. coli lysate.Purification of the HB-C2A target protein from the lysate was achievedusing a heparin sepharose column. Elution of the HB-C2A target proteinwas monitored by 280 nm absorbance of the HB affinity tag. Proteinelution was administered using a stepwise gradient of NaCl dissolved in10 mM tris-HCl buffer (pH 7.2-8.0) according to Table III.

TABLE III Stepwise NaCl Elution Gradient 100 mM 250 mM 350 mM 500 mM 750mM 1000 mM  1500 mM FIG. 4 illustrates the elution profile of bound and unbound fractions inresponse to the NaCl gradient. Results of SDS-PAGE showed that most ofthe proteins in the soluble portion of the E. coli lysate eluted as anunbound fraction in low NaCl (≧100 mM) concentration. HB-C2A bound tothe heparin stationary phase consistently eluted at 500 mM NaCl and wasmore than ˜95% pure (as assessed by staining of the SDS-PAGE gelsindependently with Coomassie blue and silver). SDS-PAGE protocol isdescribed below.

Further, the gel containing the fusion proteins was subjected to blottransfer for Western blot and dot blot analyses. Western blot and dotblot protocol, including generation of HB-antibodies, is describedbelow. FIG. 5 illustrates results of the Western blotting with lanesidentified in Table IV.

TABLE IV Western Blot Lane Identification Lane Identification 1 Proteinmarker 2 HB-C2A whole lysate 3 HB-C2A insoluble pellet 4 Purified HB-C2A5 Purified HB-S100A13* *Provided as an additional example of targetprotein bound to HB affinity tagFIG. 6 illustrates results of the dot blot analysis. As illustrated inFIG. 6, HB-C2A was detectable in amounts less than 10 ng.

Fractions containing the HB-C2A target protein were desalted by constantdialysis against 10 mM phosphate buffer (pH 6.5) containing 100 mM NaCl.The protein sample was concentrated down to 2-3 mL, and cleavage of theHB-tag from the C2A target was achieved by incubation of the HB-C2Atarget protein with one unit of thrombin per 25 μg of target protein at37° C. for 20-24 hours in 10 mM tris-HCL containing 100 mM NaCl. Thethrombin-induced cleavage reaction was stopped by the addition of 0.2Mphenylmethyl sulfonylfluoride (PMSF, 1 μL per mL of the reactionmixture). The cleavage products of the thrombin reaction were separatedby passing them through a 2-mL heparin sepharose spin column. The targetC2A protein was eluted in the wash buffer [10 mM tris-HCl (pH 7.2)containing 100 mM NaCl]. The bound HB affinity tag eluted in 10 mMtris-HC1 (pH 7.2) buffer containing 500 mM NaCl and was 95% pure.

Characterization

MALDI-MS was performed on the purified target protein and the HBaffinity-tag to check both their purity and their molecular weights. Allmass spectrometry experiments were acquired at the Statewide MassSpectrometry facility located at the University of Arkansas,Fayetteville. MALDI mass results provided in FIG. 7 displayed theexpected mass for both the HB affinity tag (Mr 4088Da) and therecombinant C2A domain (Mr 15987Da).

Isothermal titration calorimetry (ITC) experiments were performed tomeasure the heparin binding affinity of the purified HB affinity tag.ITC experiments were performed using the iTC200 (MicroCal Inc.,Northampton, Mass.) at 25° C. The HB-tag, in 10 mM phosphate buffer (pH7.2) containing 100 mM NaCl, was centrifuged to remove possible waterinsoluble material and later degassed before titration. Concentrationsof heparin to HB-tag were maintained at a ratio of 10:1. The contents ofthe syringe (heparin) were added sequentially in 1.3 μl aliquots to thecell (HB-tag) with a 12 sec interval. The raw ITC data were analyzedusing Origin Version 7.0 software provided by the vendor (MicroCal Inc.Northhampton, Mass.). Heats of reaction per injection (μcalories/sec)were determined by the integration of peak areas. Thermodynamic (ΔG, ΔH,and ΔS) and binding parameters [dissociation constant (Kd) and bindingstoichiometry (n)] were calculated by fitting the raw data to a one-siteof binding model available in Origin 7.0. ITC results demonstrated thatHB affinity tag has a nanomolar binding affinity for heparin (K_(d)˜190nM) and the HB-heparin binding proceeds with the evolution of heat(ΔH=−1.158E⁵) suggesting that the interaction is largely electrostatic.The binding stoichiometry between HB affinity tag and heparin was 1:1.FIG. 8 is an isothermogram depicting the titration of the HB affinitytag with heparin. The upper panel illustrates the raw isothermogram andthe lower panel is the best-fit binding curve using the multiple sitebinding model provided by GE Healthcare, Inc.

Far UV circular dichroism (CD) spectra were acquired using a Jasco J720spectropolarimeter. CD data were acquired at 25° C. using a 0.1 mmpathlength quartz cell. CD experiments were performed by dissolving theprotein/peptide samples (˜150 μM) in 10 mM phosphate buffer (pH 7.0)containing 100 mM NaCl. All CD spectra are an average of 10 scansacquired with a scan speed of 50 nm/second. All CD data were correctedfor background absorbance. As shown in FIG. 9, the far UV circulardichroism (CD) spectrum of the purified recombinant C2A domain showed anegative ellipticity centered at 218 nm suggesting that the backbone ofthe protein is predominantly in a helical conformation. The far UV CDspectrum of the purified recombinant C2A domain also showed a perfectmatch with the published spectrum of the C2A domain indicating that thepurified protein is in its native conformation. The far UV spectrum ofpurified HB affinity tag (obtained after thrombin cleavage ofrecombinant HB-C2A) of FIG. 10 displayed a negative CD band at around202 nm suggesting that it is in a disordered conformation. In thepresence of heparin, however, the HB affinity tag underwent a majorbackbone conformational change from a disordered state to a helicalconformation. The backbone conformational change is reflected in theappearance of the helix-characteristic double minima, located at 208 nmand 222 nm, in the far UV CD spectrum of the HB affinity tag obtained inthe presence of heparin. The heparin-induced disordered state to helixtransition plausibly suggests that the HB affinity tag can bind toheparin in the disordered state(s) and this property can be successfullyexploited to recover and purify HB-tagged recombinant proteins frominclusion bodies.

HB-antibodies (HB-Ab) for Western blotting were generated by Genescript,N.J., USA per submitted specifications. The segment of the HB tagpossessing the highest antigenicity was designed using the OptimumAntigen design program at the vendor's site(http://www.genscript.com/PolyExpress.html). HB-Ab was raised in rabbitsagainst the HB peptide sequence of SEQ ID NO:10. The cysteine residuewas placed at the C-terminal end of the peptide to enhance itsantigenicity. The designed synthetic peptide served as an efficientantigen and the antibodies raised against the peptide showed a very hightiter value. The detailed protocol for the generation of HB-Ab can beobtained at the vendor's website(http://www.genscript.com/PolyExpress.html).

Purified HB-C2A was resolved on 15% SDS-PAGE under reduced conditions.The gel containing the HB-C2A was subjected to blot transfer for Westernblot and in case of dot blot the proteins were directly spotted onto themembrane. Blotted/spotted nitrocellulose membrane was blocked using 5%skim milk in 1×TBS-T (10 mM tris, 100 mM NaCl, 0.05% Tween-20; pH 7.4)at room temperature for 1 hr. Subsequently, the membrane was washed with0.2% BSA in 1×TBS-T and the primary antibodies, raised in rabbit againstthe HB tag, was added at 1:2500 dilution and incubated overnight at 4°C. Secondary antibody conjugated with alkaline phosphatase, whichdetects the IgG rabbit antibody, was added to the membrane at 1:2500dilution and incubated for 2 hours at 4° C. The band on the membrane wasdetected using the NBT/BCIP (Nitro-bluetetrazolium/5-Bromo-4-chloro-3-indolyl phosphate) substrate within 60seconds of exposure.

IV. paration Media and Methods of Use

In a further aspect, separation media are described herein which can beused in the purification of protein compositions. A separation mediumdescribed herein comprises a support phase and a stationary phaseattached to the support phase, the stationary phase including an aminoacid sequence comprising a repeating amino acid unit of BXXXBXX, whereinB is an amino acid selected from the group consisting of histidine,lysine and arginine and X is an amino acid selected from the groupconsisting of amino acids other than histidine, lysine and arginine. Theamino acid unit can be repeated at least three times in the amino acidsequence. In some embodiments, for example, the amino acid sequenceincludes the sequence of SEQ ID NO:1 or SEQ ID NO:2. In otherembodiments, the stationary phase can include an amino acid sequenceselected from Table I. The amino acid sequence of the stationary phasecan be operable to selectively bind heparin.

The amino acid sequence of the stationary phase can be attached to thesupport phase through any suitable spacer not inconsistent with theobjectives of the present invention. In some embodiments, a spacercomprises a hydrocarbon section. A hydrocarbon section can be a linearor branched C₄ to C₂₀ hydrocarbon. Further, the spacer can comprises oneor more functionalities for reacting with the N-terminal and/orC-terminal ends of the stationary phase amino acid sequence. Forexample, the spacer can be activated by treatment withN-hydroxysuccinimide to form a stable amide linkage with the N-terminalend of the stationary phase amino acid sequence. In some embodiments,suitable support for attaching the amino acid sequence of the stationaryphase is commercially available from GE Healthcare Life Sciences asNHS-activated Sepharose 4 Fast Flow. Alternatively, in some embodiments,the amino acid sequence of the stationary phase is attached directly tothe support without an intervening spacer.

Employing a stationary phase comprising an amino acid sequence describedherein can render the separation media operable for purification ofheparin. The amino acid sequence, for example, can selectively bindheparin from a mixture of other species. The bound heparin fraction canbe subsequently eluted with a salt gradient as detailed in Section IIIand Table II herein. For example, beef-lung and porcine intestinalmucosa are rich sources of heparin. The filtered tissue homogenates canbe subjected to several cycles of ammonium sulfate treatment(s) toselectively eliminate proteins present in the tissue homogenates. Thesupernatant(s) obtained after several cycles of ammonium sulfatetreatment, are dialyzed against 10 mM phosphate buffer containing 50 mMNaCl. Heparin present in the dialyzed supernatant is can be purified ona separation medium wherein NHS Sepharose 4 Fast Flow affinity matrix ismodified or tagged with an amino acid sequence described herein, such asSEQ ID NO:1 or SEQ ID NO:2. NaCl salt gradient is used to elute thebound heparin fraction and elution monitored by 232 nm absorbance.

Sequence Listing Free Text

Designed amino acid sequence to act as an affinity tag for bindingheparin.

Designed amino acid sequence to act as an affinity tag for bindingheparin.

Designed nucleic acid for coding the amino acid sequence of SEQ ID No.:1.

Designed nucleic acid for coding the amino acid sequence of SEQ ID No.:2.

Designed amino acid sequence to act as an affinity tag for bindingheparin.

Designed amino acid sequence to act as an affinity tag for bindingheparin.

Designed nucleic acid for coding the amino acid sequence of SEQ ID No.:5.

Designed nucleic acid for coding the amino acid sequence of SEQ ID No.:6.

Designed amino acid sequence to produce polyclonal antibodies against anaffinity tag for binding heparin.

1. An affinity tag for protein purification comprising: an amino acidsequence including a repeating amino acid unit of BXXXBXX, wherein B isan amino acid selected from the group consisting of histidine, lysineand arginine and X is an amino acid selected from the group consistingof amino acids other than histidine, lysine and arginine.
 2. Theaffinity tag of claim 1, wherein the affinity tag binds heparin.
 3. Theaffinity tag of claim 1, wherein amino acid sequence comprises thesequence of SEQ ID NO:1.
 4. The affinity tag of claim 1, wherein aminoacid sequence comprises the sequence of SEQ ID NO:2.
 5. A recombinantprotein composition comprising a first amino acid sequence of a targetprotein and an affinity tag coupled to the first amino acid sequence,the affinity tag comprising an amino acid sequence for binding heparin.6. The recombinant protein composition of claim 5 further comprising aprotease cleavage site between the target protein amino acid sequenceand the affinity tag amino acid sequence.
 7. The recombinant proteincomposition of claim 6, wherein the protease cleavage site is a serineprotease cleavage site or cysteine protease cleavage site.
 8. Therecombinant protein composition of claim 7, wherein the serine proteaseis thrombin.
 9. The recombinant protein composition of claim 7, whereinthe cysteine protease is a tobacco etch virus (TEV) protease.
 10. Therecombinant protein composition of claim 5, wherein the affinity tagamino acid sequence comprises a repeating amino acid unit of BXXXBXX,wherein B is an amino acid selected from the group consisting ofhistidine, lysine and arginine and X is an amino acid selected from thegroup consisting of amino acids other than histidine, lysine andarginine.
 11. The recombinant protein composition of claim 10, whereinthe amino acid unit is repeated at least three times in the affinity tagamino acid sequence.
 12. The recombinant protein composition of claim 5,wherein the affinity tag amino acid sequence comprises the sequence ofSEQ ID NO:1.
 13. The recombinant protein composition of claim 1, whereinthe affinity tag amino acid sequence comprises the sequence of SEQ IDNO:2.
 14. A recombinant DNA molecule encoding for a protein comprising afirst amino acid sequence of a target protein and an affinity tag aminoacid sequence for binding heparin.
 15. The recombinant DNA molecule ofclaim 14, wherein the protein further comprises a protease cleavage sitebetween the target protein amino acid sequence and the affinity tagamino acid sequence.
 16. The recombinant DNA molecule of claim 14,wherein the affinity tag amino acid sequence comprises a repeating aminoacid unit of BXXXBXX, wherein B is an amino acid selected from the groupconsisting of histidine, lysine and arginine and X is an amino acidselected from the group consisting of amino acids other than histidine,lysine and arginine.
 17. The recombinant DNA molecule of claim 16,wherein the amino acid unit is repeated at least three times in theaffinity tag amino acid sequence.
 18. The recombinant DNA molecule ofclaim 14 comprising the nucleotide sequence of SEQ ID NO:3.
 19. Therecombinant DNA molecule of claim 14 comprising the nucleotide sequenceof SEQ ID NO:4.
 20. A method of purifying a recombinant proteincomposition comprising: providing a mixture including the recombinantprotein composition, the recombinant protein composition comprising afirst amino acid sequence of a target protein and an affinity tagcoupled to the first amino acid sequence, the affinity tag including anamino acid sequence for binding heparin; loading the mixture onto aseparation column having a stationary phase comprising heparin; bindingthe recombinant protein to heparin of the stationary phase; and elutingthe recombinant protein from the separation column. 21-52. (canceled)